1. Field of the Invention
The invention relates generally to compositions and methods for identifying agents that selectively bind a pentameric ligand-gated ion channel, and more specifically to an acetylcholine binding protein and to soluble pentameric ligand-gated ion channel ligand binding domains; to fluorescence-based screening assays useful for identifying agents that selectively bind acetylcholine binding proteins and/or acetylcholine receptors; and to methods of detecting agents that selectively bind a neuronal-type acetylcholine receptor.
2. Background Information
Pentameric ligand-gated ion channels (LGICs), including, for example, nicotinic acetylcholine receptors (nAChRs), comprise a superfamily of neurotransmitter receptors that allow communication between cells of the central nervous system (CNS) by converting a chemical signal, in the form of a neurotransmitter released by a cell, into an electrical signal that propagates across a target cell membrane. In neuronal signaling, depolarization of adjacent regions of the neuronal membrane allows action potentials to travel down the length of the nerve cell axons as electric signals, resulting in the rapid transmission of nerve impulses over long distances. The arrival of an action potential at the terminus of a neuron typically signals the release of neurotransmitters (e.g., acetylcholine), which carry signals between cells at a synapse. Neurotransmitters released from presynaptic cells bind to receptors on the membranes of postsynaptic cells, where they act to open pentameric LGICs. The binding of acetylcholine to an acetylcholine receptor (AChR), for example, induces a conformational change in the receptor that results in an opening of a receptor-associated ion channel, allowing the passage of charged ions across the cell membrane.
The nAChRs, like all pentameric LGICs, are multisubunit proteins that mediate synaptic transmission between nerve cells, and between nerve and muscle cells, upon interaction with the neurotransmitter acetylcholine. The nAChRs, which include neuronal-type nAChRs and muscle-type nAChRs, contain five subunits that are arranged as a cylinder in the cell membrane. The nAChRs are present in a variety of tissues and control skeletal muscle contraction, and sympathetic and parasympathetic ganglia function, thereby controlling cardiovascular and visceral functions, and are important in communication pathways in the brain. These receptors are disturbed in patients with Alzheimer's disease, Parkinson's disease, schizophrenia and other disorders involving memory loss, cognitive problems and dementia. In addition, neuronal-type nAChRs are the involved in nicotine addiction.
Due to their pervasive role in CNS function, pentameric LGICs such as nAChRs provide important targets for drugs. For example, nAChR modulators are used to reduce blood pressure, and are used in surgery as neuromuscular blockers, where modulators function as competitive agonists or depolarizing agents. As such, nAChR modulating drugs have many pharmacological actions, and synthetic compounds are being examined for efficacy in a number of therapeutic indications, including, for example, in treating Alzheimer's disease, Parkinson's disease, nicotine addiction, epilepsy, attention deficit disorder and pain, and as neuroprotective agents (see, e.g., “Neuronal Nicotinic Receptors: Pharmacology and Therapeutic Opportunities”, Eds. Arneric and Brioni (Wiley-Liss, Inc. 1999)).
Despite the importance of nAChR and other pentameric LGICs in nervous system function and the role of the receptors in many diseases, only a limited number of drugs are available for modulating pentameric LGIC activity. One problem in identifying agents that can selectively modulate LGIC activity is that the pentameric LGICs comprise transmembrane bound proteins, which are not readily adaptable to solution based screening assays. Thus, a need exits for screening assays that conveniently can be used to identify agents that can selectively bind a pentameric LGIC and act, for example, as an agonist or as an antagonist of the LGIC function.